Molecular characterization of a new excitatory insect neurotoxin with an analgesic effect on mice from the scorpion Buthus martensi Karsch.

Besides the neurotoxins active on mammals, a new excitatory insect selective toxin with a mice analgesic activity was found and purified from the venom of the scorpion Buthus martensi Karsch (BmK) (Ji, Y.H., Mansuelle, P., Terakawa, S., Kopeyan, C., Yanaihara, N., Hsu, K., Rochat, H., 1996. Toxicon 34, 987; Luo, M.J., Xiong, Y.M., Wang, M., Wang, D.C., Chi, C.W., 1997. Toxicon 35, 723.). This peptide (designated as BmK IT-AP) is composed of 72 amino acid residues. Its primary structure was determined by automated Edman degradation of the N-terminal part of the reduced and S-carboxamidemethylated protein and its lysylendopeptidase degraded fragments. Based on the determined sequence, the gene specific primers were designed and synthesized for 3' and 5' RACE (rapid amplification of cDNA ends). Their partial cDNA fragments obtained by 3' and 5' RACEwere cloned and sequenced and the full length cDNA sequence of BmK IT-AP was then completed by overlapping their two partial cDNA sequences. It encodes a precursor of 90 amino acid residues: a signal peptide of 18 residues and a mature peptide of 72 residues which are consistent with the determined protein sequence of BmK IT-AP. The genomic DNA of the peptide was also amplified by PCR from the scorpion genomic DNA and sequenced, which is a first report on the genomic structure of a scorpion toxin specific for insects. Its sequence revealed an intron of 590 bp inserted in the end part of the signal peptide. The peptide caused a fast excitatory contraction paralysis on house fly larvae. Furthermore, the peptide also showed an obvious analgesic effect on mice, as assayed by using a twisting test model. This effect of BmK IT-AP well characterized at molecular level is first reported among the known scorpion insect neurotoxins.     

The cDNA sequence of an excitatory insect selective neurotoxin from the scorpion Buthus martensi Karsch.

The full-length cDNA of an excitatory insect selective neurotoxin was amplified from total cDNAs of venomous glands of the scorpion Buthus martensi Karsch (BmK) using the 3'RACE and 5'RACE (rapid amplification of cDNA ends, RACE) method and sequenced. The cDNA encoded a precursor of the insect toxin of 88 amino acid residues, including a signal peptide of 18 residues and a mature toxin of 70 residues. The cDNA deduced sequence of this toxin was homologous with the determined amino acid sequence of BmK IT1, an excitatory insect toxin purified from the scorpion venom, except for three different residues, two at the positions 24-25, and another in the COOH-terminus of the toxin. Among them the COO-terminal residue Gly in the cDNA deduced sequence was predominantly different from the conserved residue Asn found in other known scorpion excitatory insect toxins.     

Gene cloning and sequencing of BmK AS and BmK AS-1, two novel neurotoxins from the scorpion Buthus martensi Karsch.

Based on the known amino acid sequences of BmK AS and BmK AS-1, the gene specific primers were designed and synthesized for 3' and 5' RACE (Rapid Amplification of cDNA Ends). Their partial cDNA fragments obtained by 3' and 5' RACE were cloned and sequenced, and the full length cDNA sequences of BmK AS and BmK AS-1 were then completed by overlapping their two partial cDNA sequences, respectively. The predicted amino acid sequences both consist of 85 amino acid residues including a putative signal peptide of 19 residues and a mature toxin of 66 residues. They are different in 17 amino acid residues, among them 11 residues in the mature toxin. The predicted amino acid sequences of BmK AS and BmK AS-1 were almost consistent with those determined and revised (personal communication), only different in one and two residues at their COO-terminal parts, respectively. Based on the determined cDNA sequences, and using the total DNAs isolated from the scorpion venom glands as a template, the genomic DNAs of BmK AS and BmK AS-1 were also amplified by PCR and sequenced. It showed that no intron was inserted in their open reading frames, while in the exon of signal peptide sequences of other Na+, K+ and Cl- channel toxins from the same scorpion, an intron is usually found. However, the Northern blot hybridization results indicated that the sizes of their mRNA should be around 800 bp. Their extra sequences around 400 bp which might function as an intron should be located at their 5' untranslated regions.     

Cloning and characterization of the cDNA sequences of two venom peptides from Chinese scorpion Buthus martensii Karsch (BmK).

From a cDNA library made from venom glands of Chinese scorpions of Buthus martensii Karsch, full-length cDNAs encoding precursors of two venom peptides have been isolated using a cDNA probe synthesized by polymerase chain reaction. Sequence analysis of the cDNAs revealed that one encoded precursor was 85 amino acid residues long including a signal peptide of 19 residues and a mature peptide (named BmK T) of 66 residues, and another encoded precursor was 84 residues long containing the same length signal peptide and a mature peptide (BmK M4 isoform, named BmK M4') of 64 residues. The analysis of amino acid sequence similarity indicated that the BmK T was homologous with both mammalian and insect toxins from BmK scorpion or other scorpions, and the BmK M4' was highly homologous with the members of the mammalian neurotoxin family of BmK, having two point mutations in amino acid residue sequence compared to BmK M4, a natural toxin from BmK.     

Nucleotide sequence and structure analysis of a cDNA encoding an alpha insect toxin from the scorpion Leiurus quinquestriatus hebraeus.

A approximately 370 base pair cDNA encoding the alpha insect toxin Lqh alpha IT of the scorpion Leiurus quinquestriatus hebraeus was cloned and sequenced. The deduced amino acid sequence for the putative mature polypeptide is identical to the protein sequence determined chemically (Eitan et al., Biochemistry 29, 5941, 1990). A 19 amino acid signal peptide precedes the 64 amino acid long toxin. Two additional amino acid residues that do not correspond to the purified toxin are found at the COOH-terminus and may imply post-translational modification. The signal peptide region in the present clone differs obviously from that encoding the depressant insect toxin LqhIT2 derived from the same venom, but strongly resembles the leader peptide sequence of an alpha-mammal toxin from the scorpion Androctonus australis.     

Identification of BmKAPi, a novel type of scorpion venom peptide with peculiar disulfide bridge pattern from Buthus martensii Karsch.

A novel cDNA sequence encoding a new type of scorpion venom peptide (BmKAPi) was first isolated from the venom gland of Buthus martensiiKarsch by cDNA library screening combined with 5′-race. The encoded precursor of BmKAPi consisted of 89 amino acid residues including a signal peptide of 24 residues, a putative mature peptide of 64 residues (BmKAPi) and an extra basic residue at the C-terminus which might be removed in the post-translational processing. BmKAPi is stabilized by five disulfide bridges, whereas all other disulfide-bridged scorpion toxins described are cross-linked by three or four disulfide bridges. It suggested the three-dimensinal scaffold of BmKAPi might be different from other scorpion toxins. The amino acid sequence of BmKAPi showed no homology with other scorpion venom peptides, but shared a little similarity with some anticoagulant peptides and proteinase inhibitors isolated from hookworm, honeybee or European frog, respectively. RT-PCR analysis showed that BmKAPi mRNA could be induced by venom extraction suggesting BmKAPi might be a component of scorpion venom. These results suggest that BmKAPi is a new type of scorpion venom peptide different from other described scorpion toxins in structural and functional aspects.     

Identification of BmKAPi, a novel type of scorpion venom peptide with peculiar disulfide bridge pattern from Buthus martensii Karsch

A novel cDNA sequence encoding a new type of scorpion venom peptide (BmKAPi) was first isolated from the venom gland of Buthus martensiiKarsch by cDNA library screening combined with 5′-race. The encoded precursor of BmKAPi consisted of 89 amino acid residues including a signal peptide of 24 residues, a putative mature peptide of 64 residues (BmKAPi) and an extra basic residue at the C-terminus which might be removed in the post-translational processing. BmKAPi is stabilized by five disulfide bridges, whereas all other disulfide-bridged scorpion toxins described are cross-linked by three or four disulfide bridges. It suggested the three-dimensinal scaffold of BmKAPi might be different from other scorpion toxins. The amino acid sequence of BmKAPi showed no homology with other scorpion venom peptides, but shared a little similarity with some anticoagulant peptides and proteinase inhibitors isolated from hookworm, honeybee or European frog, respectively. RT-PCR analysis showed that BmKAPi mRNA could be induced by venom extraction suggesting BmKAPi might be a component of scorpion venom. These results suggest that BmKAPi is a new type of scorpion venom peptide different from other described scorpion toxins in structural and functional aspects.     

Characterisation of the gene encoding the alpha-toxin Amm V from the scorpion Androctonus mauretanicus mauretanicus.

The full-length cDNA encoding the scorpion alpha-toxin Amm V was amplified from a cDNA library produced from the venom glands of the scorpion Androctonus mauretanicus mauretanicus from Morocco. We deduced the amino acid sequence of the encoded precursor protein and found that the mature toxin was similar to the previously characterised toxin. The genomic DNA sequence encoding the toxin was also amplified, subcloned and sequenced. This also led to the isolation of a new Amm V related-gene. Then, for the first time, we studied changes in the level of toxin mRNA synthesis over time.     

Molecular cloning and sequence analysis of cDNAs encoding a beta-toxin-like peptide and two MkTx I homologues from scorpion Buthus martensii Karsch.

Three full-length cDNAs, one encoding the precursor of a beta-toxin-like peptide (named BmKBT) and the other two encoding those of (MkTx I) homologues (named MkTx II and MkTx III, respectively), were isolated from a venom gland cDNA library of the Chinese scorpion Buthus martensii Karsch, by screening with a cDNA fragment generated by PCR. The encoded precursor of BmKBT contained 83 amino acid residues including a signal peptide of 19 residues, a mature peptide of 63 residues and an extra basic residue (Lys) which have to be removed in the processing step. The deduced amino acid sequence of BmKBT showed 52% homology to that of beta-neurotoxin TsVII isolated from scorpion Tityus serrulatus. However, the positions of disulfide bridges have a little variation between the two peptides. The precursors of MkTx II and MkTx III both contained 85 amino acid residues including a signal peptide of 19 residues, a mature peptide of 64 residues and two extra residues (Gly-Arg) which have to be removed in the processing step, too. There was high sequence similarity (90%) between the two peptides. The sequences of mature MkTx II and MkTx III were highly homologous with MkTx I isolated from scorpion Buthus martensii Karsch, both showing 90% identities.     

Precursor nucleotide sequence and genomic organization of BmTXKS1, a new scorpion toxin-like peptide from Buthus martensii Karsch.

Scorpion venom contains a variety of small peptides, which can modulate Na+, K+, Ca2+ and Cl- channel conductance in excitable and non-excitable tissues. A novel full-length cDNA encoding a new toxin-like peptide (named BmTXKS1) was isolated from the venom gland cDNA library of Buthus martensii Karsch. The precursor consists of 60 amino acid residues, with a putative signal peptide of 28 residues and an extra residue, and a mature peptide of 31 residues with an amidated C-terminal. BmTXKS1 shared close homology with BmP01 in 5'UTR and the region encoding the putative signal peptide; especially, the positions of six cysteines are highly conserved among BmTXKS1, PbTX1 and P01-type subfamily of scorpion K+ channel toxins, suggesting that they all should present a common three-dimensional fold, namely the Cysteine-Stabilized alphabeta(CSalphabeta) motif. By PCR amplification of the genomic region encoding BmTXKS1, we have confirmed the identity of our cloned cDNA, and found that BmTXKS1 gene contains an intron, which is completely identical with that of the characterized scorpion K+-channel-ligands in the size, consensus junctions, putative branch point and A+T abundance.     

A novel scorpion toxin blocking small conductance Ca activated K channel

Small conductance calcium activated potassium channels (SK) are crucial in the regulation of cell firing frequency in the nervous system and other tissues. In the present work, a novel SK channel blocker, designated BmSKTx1, was purified from the scorpion Buthus martensi Karsh venom. The sequence of the N-terminal 22 amino acid residues was determined by Edman degradation. Using this sequence information, the full-length cDNA and genomic gene of BmSKTx1 were cloned and sequenced. By these analyses, BmSKTx1 was found to be a peptide composed of 31 amino acid residues with three disulfide bonds. It shared little sequence homology with other known scorpion -KTxs but showed close relationship with SK channel blockers in the phylogenetic tree. According to the previous nomenclature, BmSKTx1 was classified as -KTx14.1. We examined the effects of BmSKTx1 on different ion channels of rat adrenal chromaffin cells (RACC) and locust dorsal unpaired median (DUM) neurons. BmSKTx1 selectively inhibited apamin-sensitive SK currents in RACC with K_d of 0.72 μM and Hill coefficient of 2.2. And it had no effect on Na⁺, Ca²⁺, Kv, and BK currents in DUM neuron, indicating that BmSKTx1 was a selective SK toxin.     

Characterisation of the genes encoding Aa1 isoforms from the scorpion Androctonus australis.

Aa1 is a toxin purified from the venom of the North African scorpion Androctonus australis. It blocks fast K(+) currents in cerebellar granular cells [Biochim. Biophys. Acta 1468 (2000) 203]. Two full-length cDNAs (about 250 bp) encoding the precursors of putative Aa1 isoforms (AaTX1 and AaTX2) were amplified by PCR from a venom gland cDNA library of A. australis. The deduced precursors were composed of 59 amino acid residues including a signal peptide of 22 residues and a mature toxin of 37 residues. The peptides display 94% sequence identity with Aa1. Intron-exon organisation of the gene corresponding to the AaTX1 cDNAs was also depicted.     

Genomic characterisation of the toxin Amm VIII from the scorpion Androctonus mauretanicus mauretanicus.

The genomic DNA sequence encoding the scorpion toxin Amm VIII was amplified from genomic DNA of the scorpion Androctonus mauretanicus mauretanicus from Morocco, subcloned and sequenced. An intron, with a high A+T content (73.5%), split a Gly codon at the end of the precursor signal peptide and the consensus GT/AG splice junction was identified in the Amm VIII gene. This intron of only 166 bp is the smallest intron described so far for a long-chain scorpion toxin gene. In addition, this study led to the identification of three new toxin-related genes. From the deduced amino acid sequences of the encoded precursor proteins, we found that the mature putative toxins were highly similar to the scorpion toxins Leiurus quinquestriatus quinquestriatus IV and Odonthobuthus doriae 1.     

Characterization of venom components from the scorpion Androctonus crassicauda of Turkey: peptides and genes.

The soluble venom from the scorpion Androctonus crassicauda was fractionated by high performance liquid chromatography. At least 44 different sub-fractions were resolved and collected for finger print mass analysis using an electrospray mass spectrometer. This analysis revealed the presence of 80 distinct molecular mass components, from which five were further characterized. A peptide, named Acra1 was fully sequenced. It contains 58 amino acid residues cross-bridged by six cysteines forming three disulfide pairs, with a molecular mass of 6497 Da. A second purified peptide named Acra2 was partially sequenced with a molecular mass of 7849 Da. Acra1 is toxic and Acra2 is lethal to mice, at the dose assayed. Additionally, a cDNA library of the venomous gland of one specimen was prepared and several clones were obtained among which is one that codes for Acra1. Three analog gene sequences were found with point mutations either in the section that corresponds to the mature peptide or to the signal peptide. The signal peptide is 22 amino acid residues long. Several other gene sequences obtained suggest the presence in this venom of three distinct groups of peptides, among which are peptides similar to known Na(+)-channel specific toxins of other scorpions. A new type of peptide was identified with odd number of cysteines (seven), allowing the formation of heterodimers with molecular masses in the range of 16,000 atomic mass units (a.m.u.).     

Molecular cloning and genomic organization of a K(+) channel toxin from the Chinese scorpion Buthus martensii Karsch.

A full-length cDNA encoding the precursor of a K(+) channel toxin (BmTX2) was first isolated from a venom-gland cDNA library of the Chinese scorpion Buthus martensii Karsch. The precursor is composed of a signal peptide of 21 residues and a mature toxin of 37 residues with three disulfide bridges. The genomic gene of BmTX2 was also cloned and sequenced. It consisted of two exons, disrupted by an intron of 81 bp inserted in the region encoding signal peptide.     

Cloning and characterization of a cDNA sequence encoding the precursor of a chlorotoxin-like peptide from the Chinese scorpion Buthus martensii Karsch.

A full-length cDNA sequence encoding the precursor of a venom peptide with homology to chlorotoxin (named BmKCT) was isolated from a cDNA library made from the venom glands of the Chinese Scorpion Buthus martensii Karsch. The encoded precursor of BmKCT was 59 amino acid residues long including a signal peptide of 24 residues and a mature toxin of 35 residues with four disulfide bridges. The sequence of BmKCT is similar (68% identities) to that of chlorotoxin isolated from Leiurus quinguestriatus quinquestriatus. BmKCT is the first report of the cDNA sequences encoding four-disulfide-bridged short-chain toxins from Buthus martensuii Karsch so far.     

Isolation and characterization of a novel P-II class snake venom metalloproteinase from Trimeresurus stejnegeri.

Stejnitin, a novel class P-II snake venom metalloproteinase (SVMP) with a molecular weight of about 35kDa, was purified from Trimeresurus stejnegeri venom. The cDNA of stejnitin encoded a polypeptide of 295 amino acid residues which comprises a signal peptide, proprotein, metalloproteinase domain, spacer and disintegrin domain. The protein sequence deduced from cDNA was confirmed by peptide mass fingerprinting analysis. It is highly homologous to the members of subclass P-IIa SVMPs which comprises metalloproteinase and disintegrin together. Results from DNA fragmentation and flow cytometry analysis also indicated that stejnitin is able to induce apoptosis of ECV304 cells (R=0.908, P=0.012).     

Cloning and molecular characterization of BmHYA1, a novel hyaluronidase from the venom of Chinese red scorpion Buthus martensi Karsch

In this communication, the full protein sequence of a novel venom hyaluronidase BmHYA1 was reported. It is the first full hyaluronidase amino acid sequence from scorpion venom. It was deduced from nucleotide sequence by 3′ Rapid Amplification of cDNA Ends (RACE) PCR cloning, followed by alignment with the N-terminal amino acid sequence, which was obtained by Edman degradation. BmHYA1 has 385 amino acid residues containing five potential N-glycosylation sites. The phylogenetic analysis indicates early divergence and independent evolution of BmHYA1 from other hyaluronidases.     

Molecular characterization of L-amino acid oxidase from king cobra venom.

An L-amino acid oxidase from Ophiophagus hannah snake venom (Oh-LAAO) was purified by successive gel filtration, ion-exchange and heparin chromatography. Oh-LAAO did not induce platelet aggregation; however, it had potent inhibitory activity on platelet aggregation induced by ADP and U46619, but showed no effect on platelet aggregation induced by thrombin, mucetin, ristocetin and stejnulxin. By RT-PCR and 5'-RACE methods, the complete Oh-LAAO cDNA was cloned from the venom gland total RNA preparations. The cDNA sequence contains an open-reading frame (ORF) of 1476-bp, which encodes a protein of 491 amino acids comprising a signal peptide of 25 amino acids and 466-residue mature protein. The predicted protein sequence of Oh-LAAO was confirmed by N-terminal and trypsin-digested internal peptides sequencing together with peptide mass fingerprinting. cDNAs encoding for ORF of LAAOs from Bungarus fasciatus and B. multicinctus were cloned and reported in this study. In addition, partial cDNA encoding for Naja atra LAAO was also reported. Oh-LAAO shared approximately 50% protein sequence identity with other known snake venom LAAOs. Phylogenetic analysis indicated that Oh-LAAO is evolutionary distant to other snake venom LAAOs.